Detection and distinguishing infections bursal disease virus (ibdv) strains by molecular biology method

ABSTRACT

The present invention relates to a novel method to detect and differentiate different strains of infectious bursal disease virus (IBDV) in a chicken and other bird sample. RNA was obtained from said samples by using a pair of primer (Primer FVVC &amp; RVVC) in a reverse transcriptase-polymerase chain reaction. Two different primer combinations (Primer IF &amp; IVIR) and (Primer IF &amp; RCLA) and real-time polymerase chain reaction conditions were designed and optimized for rapid differentiation of very virulent and vaccine strains of IBDV based on detection of signatory threshold cycle (Ct) and melting temperature (Tm) values.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.11/268,341, filed Nov. 7, 2005, and now pending, the entire disclosureof which is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the detection and distinguishing InfectiousBursal Disease Virus (IBDV) strains by a Molecular Biology Method inchicken or other birds. More particularly, this invention relates todistinguishing different Infectious Bursal Disease Virus (IBDV) strainsin chicken and other bird sample by Real-time Polymerase Chain Reaction(PCR) method.

BACKGROUND OF THE INVENTION

Infectious bursal disease (IBD) is an acute contagious viral disease ofyoung chickens often known as Gumboro disease (Kibenge et al., J GenVirol. 69(Pt 8):1757-1775, 1988; Lasher et al., Avian Dis. 41(1):11-19,1997). The etiological agent, IBD virus (IBDV), has a predilection forthe cells of the bursa of Fabricius where the virus infects activelydividing and differentiating lymphocytes of the B-cell lineage(Burkhardt et al., Arch Virol. 94(3-4):297-303, 1987). Thus, IBD is afatal immunosuppressive disease causing heavy losses to the poultryindustry (Eterradossi et al., Arch Virol. 143(8):1627-1636, 1998).

The first outbreak of IBDV was reported in commercial chicken flocks inDelaware, USA (Cosgrove, Avian Dis. 6:385-389, 1962). The IBDV strains,which were isolated during the outbreak, now referred to as classicalserotype I isolates. The disease was also first report in Europe in 1962(Faragher, Vet. Bull. 42:361-369, 1972). And from 1966 to 1974, IBD wasreported in the Middle East, Southern and Western Africa, India, the FarEast and Australia (Faragher, 1972; Firth, Aust Vet J. 50(3):128-130,1974; Jones, N Z Vet J. 34(3):36, 1986; van den Berg, Avian Pathol.29:175-194, 2000). In most cases, the IBDV strains that associated withthe outbreaks were of low virulence and caused only 1 to 2% of specificmortality (van den Berg, 2000).

However, a new IBDV strain (antigenic variant) emerged and able to causeup to 5% specific mortality in USA (Rosenberger and Cloud, Avian Dis.33(4):753-759, 1989). The antigenic variant was recovered from flockswith selection pressure of field vaccination against classical IBDVserotype I (Snyder, 1990). Although being antigenic variant theseisolates have only minor amino acid changes and do not form a separateserotype.

Nevertheless, these changes occur at the VP2 conformation-dependentantigenic epitopes that are responsible for stimulating virusneutralizing antibodies (Bayliss et al., J Gen Virol. 71(Pt6):1303-1312, 1990). Currently, variant form of IBD has been reportedoutside Central America particularly in countries such as China (Cao etal., Avian Dis. 42(2):340-351, 1998), South America (Banda et al., AvianDis. 47(1):87-95, 2003) and Australia (Sapats and Ignjatovic, ArchVirol. 145(4):773-785, 2000).

Since variant IBDV causes only changes at the bursa and depending on theimmune status of the chickens, the disease is often manifested withsubclinical signs, it is difficult to detect variant IBDV in commercialflocks. Hence, variant IBDV may be common in many countries in the worldbut remains undiagnosed. A second serotype—serotype II of IBDV wasidentified in 1987 (McNulty and Saif, Avian Dis. 132(2):374-375, 1988).Serotype II IBDV isolates are apathogenic and are recovered mainly fromturkeys (Ismail et al., Avian Dis. 32(4):757-759, 1988).

In the 1990s, IBDV isolates, which were able to break through levels ofmaternal antibodies that normally were protective, were reported inEurope (Chettle et al., Vet Rec. 125(10):271-272, 1989). These isolates,the so called very virulent IBDV are causing more severe clinical signsduring an outbreak which mortality approaching 100% in susceptibleflocks, and are now found almost world-wide (van den Berg, 2000). Theemergence of very virulent strains of IBDV has complicated theimmunization programs against the disease.

Early vaccination may result in failure due to interference with thematernal antibody, whilst its delay may cause field virus infections.Currently, outbreaks of vvIBDV have been reported throughout variouscountries in the world (Banda et al., Avian Dis. 47(1):87-95, 2003; Caoet al., Avian Dis. 42(2):340-351, 1998; Chai et al., Arch Virol.146(8):1571-1580, 2001; Chettle et al., 1989; Eterradossi et al.,Zentralbl Veterinarmed B. 39(9):683-691, 1992; Hoque et al., J BiochemMol Biol Biophys. 6(2):93-99, 2002; Liu et al., Virus Genes.24(2):135-147, 2002; Majo et al., Avian Dis. 46(4):859-868, 2002; Ruddet al., Aust Vet J. 81(3):162-164, 2003; Scherbakova et al., 1998; Ture,et al., Avian Dis. 42(3):470-479, 1998; Zorman-Rojs et al., Avian Dis.47(1):186-192, 2003).

In designing an effective disease control program one should considerthe diagnostic methods use to diagnose disease caused by infectiousagent. Currently, IBD can be diagnosed based on virus isolation,electron microscopy, immunofluorescence, virus neutralization,monoclonal antibody assays, and/or enzyme-linked immunosorbent assay(Jackwood et al., Clin Diagn Lab Immunol. 3(4):456-463, 1996; Jackwoodet al., Avian Dis. 40(2):457-460, 1996; Liu et al., J Virol Methods.48(2-3):281-291, 1994; Lukert and Saif, Infectious bursal disease. In:Diseases of Poultry, 10th edn (Eds. Calnek et al.), Iowa StateUniversity Press, Ames, Iowa, pp. 721-738, 1997; Wu et al., Avian Dis.36(2):221-226, 1992). However, these methods have one or moredisadvantages such as time consuming, labour intensive, expensive and oflow sensitivity (Wu et al., 1992).

Recently, the reverse transcriptase polymerase chain (RT-PCR) has beenused to detect IBDV based on the amplification of the centralhypervariable region of the VP2 region (Tham et al., J Virol Methods.53(2-3):201-212, 1995; Jackwood and Nielsen, Avian Dis. 41(1):137-143,1997). Subsequently, RT-PCR assay followed by restriction fragmentlength polymorphism (RFLP) also has been used to detect anddifferentiate IBDV strains (Jackwood and Sommer, Avian Dis.41(3):627-637, 1997; Jackwood and Sommer, Avian Dis. 43(2):310-314,1999; Hoque et al., Avian Pathol. 30:369-380, 2001; Ture et al., AvianDis. 42(3):470-479, 1998; Zierenberg et al., 2001).

Although, this method able to differentiate different IBDV strains, itis not automated and time consuming. Both radioactive andnon-radioactive based nucleic acid probes that can differentiate IBDVstrains have been used in the detection of IBDV (Akin et al., Vet DiagnInvest. 5(2):166-173, 1993; Davis and Boyle, Avian Dis. 34(2):329-335,1990). However, apart for academic interest, their use in diagnosing IBDis uncommon.

Fluorescence-based real-time PCR assays have been developed to provide arapid and sensitive method for quantifying nucleic acids (Gibson et al.,Genome Res. 6(10):995-1001, 1996; Heid et al., Genome Res.6(10):986-994, 1996; Desjardin et al., J Clin Microbiol.36(7):1964-1968, 1998). In this assay, reactions are monitored by thepoint in time during cycling when amplification of a PCR product isfirst detected rather than the amount of PCR product accumulated after afixed number of cycles. There are currently 2 general approaches inreal-time PCR depending on the types of fluorescence dyes. The simplestmethod uses fluorescent dye, SYBR Green I that bind specifically todouble stranded DNA (Morrison et al., Biotechniques. 24(6):954-958, 960,962, 1998). The major problem with SYBR Green I-based detection is thatnon-specific amplifications cannot be distinguished from specificamplifications. However, specific amplification can be verified bymelting curve analysis (Ririe et al., Anal Biochem. 245(2):154-160,1997).

The other dyes (TaqMan, Molecular Beacons, Scorpion) rely on thehybridization of fluorescence labeled probes to the correct amplicon(Wittwer et al., Biotechniques. 22(1):130-131, 134-138, 1997; Bonnet etal., 1999). Accumulation of PCR products is detected by monitoring theincrease in fluorescence of the reporter dye. The threshold cycle (Ct)is defined as the fractional cycle number at which the reporterfluorescence generated by the accumulating amplicons passes a fixedthreshold above baseline (Mackay et al., Nucleic Acids Res.30(6):1292-1305, 2002).

Hence, a plot of the log of initial target copy number for a set ofstandards versus Ct is a straight line whereby the higher the startingcopy number of the nucleic acid target, the sooner a significantincrease in fluorescence is observed (Higuchi et al., Biotechnology(NY). 11(9):1026-1030, 1993; Gibson et al., 1996; Heid et al., 1996;Desjardin et al., 1998). It has also been established that primerscombination playing an important role for the Ct value prediction, wherethe approach is similar to the analysis of single-nucleotidepolymorphism (Frederique et al., 2003; Christy et al., 2002; Srinivas etal., 2000). Thus, several recent studies have used SYBR Green I basedreal-time PCR to differentiate different serotypes or strains oforganisms based on Ct and/or Tm values (Aldea et al., J Clin Microbiol.40(3):1060-1062, 2002; Beuret, J Virol Methods. 115(1):1-8, 2004;Nicolas et al., J Microbiol Methods. 51(3):295-299, 2002; Shu et al., JClin Microbiol. 41(6):2408-2416, 2003). A quantitative real-time PCRassay based on TaqMan has been developed to detect IBDV (Moody et al., JVirol Methods. 2000 85(1-2):55-64, 2000). In other recent studies byJackwood and Sommer (Virology. 304(1):105-113, 2002) and Jackwood et al.(Avian Dis. 47(3):738-744, 2003), TaqMan based real-time PCR was shownto be able to detect vaccine and wild type IBDV strains in infectedchickens. However, the assay is expensive and more complex compared tothe method established in this study. In addition, the application ofthe method to differentiate very virulent and vaccine strains IBDV isnot known.

SUMMARY OF THE INVENTION

The biological material, which is to be investigated, was obtained insome suitable matter and isolated. By means of standardized methods, RNAwas isolated from the material. A defined amount of RNA was transcribedinto cDNA by means of conserved primers in RT reaction. Subsequently, adefined amount of cDNA was used to generate specific products using PCR.

One of the most common dyes that bind to double-stranded DNA that iscommonly used in real-time PCR is SYBR Green I. In real-time PCR,measurements are detected during the exponential phase of the reactiontypically by obtaining the threshold cycle (Ct) value. Ct value can bedefined as the fractional cycle number at which there is a significantincrease in fluorescence above a specified threshold.

The Ct value is also proportional to the numbers of target copiespresent in the samples. In SYBR Green I based real-time PCR thespecificity of the amplification is determined by measuring the meltingtemperature (Tm) of the product in melting curve analysis. DifferentIBDV strains can be differentiated based on the Ct values by usingdifferent primer combinations and the detection of expected Tm confirmthe specificity of the amplification.

The PCR conditions are optimized in order to obtain effective PCRparameters on the ingredients and profiles using samples containing IBDVRNA in a SYBR Green I based real-time PCR. Hence, for differentiation ofvery virulent from vaccine strains of IBDV by using Primer IF & IVIR andPrimer IF & RCLA, a PCR product from very virulent strain IBDV has anearly amplification (Ct value between 19 to 28 and Tm between 86° C. to88° C.) and late amplification (Ct value>29 and Tm<82° C.) or noamplification (Ct value 0 and Tm<82° C.), respectively.

Meanwhile, differentiation of vaccine from very virulent strains of IBDVby using Primer IF & IVIR and Primer IF & RCLA, the PCR product fromvaccine strain of IBDV has an early amplification (Ct value between 19to 28 and Tm between 86° C. to 88° C.) and late amplification (Ctvalue>29 and Tm<82° C.) or no amplification (Ct value 0 and Tm<82° C.),respectively.

It is an objective of the invention to provide a new method fordifferentiating IBDV strains and for identifying IBDV strains based onthe detection of signatory Ct and Tm values.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts nucleotide sequences and the deduced amino acidstranslation of primers IVIR and RCLA. The IVIR primer matched to veryvirulent strains meanwhile primer RCLA matched to vaccine strains. ^(a)The nucleotide sequences of the primers that associated with amino acidvariations are bold. ^(c) The primer conserved to very virulent strains,UPM97/61 (AF247006), UPM94/273 (AF527039), OKYM (D49706), UK661(X92760), IBDKS (L42284), D6948 (AF240686), BD3/99 (AF362776), Tasik94(AF322444), Chinju (AF508176), HK46 (AF092943), SH95 (AF13474), Gx (AY444873), SDH1 (AY323952) and TO9 (AY099456). ^(d) The primer conservedto vaccine (attenuated) strains, D78 (AF499929), Cu-1M (AF362771), P2(X84034), CT (AJ310185), CEF94 (AF194428), PBG-98 (D00868), JD1(AF321055), HZ-2 (AF321054) and Edgar (AF462026).

FIG. 2A shows the influence of the number of cycles in real-time PCRassay using template from vaccine strain D78 and mismatch primercombinations. FIG. 2B shows evidence of faint PCR product of theexpected size (316 bp) (arrow) and primer-dimer from amplification ofD78 cDNA with mismatch primer combinations (IF & IVIR) after 33 cycles.The Tm of the mismatch product was 86° C. to 87° C. whilst the Tm forthe dimer product was 77° C. Lane M, 100 bp marker (Promega, USA), Lane1, 2 and 3 are triplicate of D78 with primers IF & IVVR.

FIG. 3 shows the performance of the real-time in detecting very virulentstrain UPM94/273. Reverse transcription was generated from 12,000 ng/μlof total RNA. The real-time PCR was performed using 10-fold dilutionsfrom 10⁰ to 10⁻⁵ (7,700 ng/μl to 0.077 ng/μl) of cDNA template(UPM94/273), match primer combination (primer IF & IVIR), mismatchprimer combination (primer IF & RCLA), 1 μl of SYBR Green I (diluted1:10³) as labeling dye and fluorescence threshold limit set at 0.01.Amplification of PCR product using match primer was detected fromundiluted until 10⁻³ diluted cDNA whilst amplification using mismatchprimer was detected only from undiluted cDNA (FIGS. 3A and 3C). Withmatch primer combinations, the Tm of amplicons from undiluted to 10⁻³diluted cDNA was between 87.2° C. to 87.6° C. whilst the Tm for 10⁻⁴ to10⁻⁵ diluted cDNA and negative control ranged from 78° C. to 78.4° C.Melting curve analysis also showed that the mismatch primer combinationsproduced a product with Tm of 87.6° C. from undiluted cDNA whilst the Tmfor 10⁻¹ until 10⁻⁵ diluted cDNA and negative control was 81.6° C. (FIG.3D). A standard curve derived from 10-fold diluted cDNA of very virulentIBDV strain indicated a linear relationship was observed between theamount of input cDNA and the Ct value. The regression equation wasCt=3.7765 (log 10 dilution)+16.393 and R²=0.999 (FIG. 3E).

FIG. 4 depicts the performance of the real-time in detecting vaccinestrain D78. Reverse transcription was generated from 6,500 to 7,000ng/μl of total RNA. The real-time PCR was performed using 10-folddilutions from 10⁰ to 10⁻⁵ (6,600 ng/μl to 0.066 ng/μl) of cDNA template(D78), match primer combination (primer IF & RCLA), mismatch primercombination (primer IF & IVIR), 1 μl of SYBR Green I (diluted 1:10³) aslabeling dye and fluorescence threshold limit was set at 0.01.Amplification of PCR product using match primer was detected fromundiluted until 10⁻³ diluted cDNA whilst amplification using mismatchdetected only from undiluted cDNA (FIGS. 4A and 4C). With match primercombinations, the products obtained from undiluted until 10⁻³ dilutedcDNA produced a Tm ranged from 86° C. to 88° C. The Tm for 10⁻⁴ to 10⁻⁵diluted cDNA and negative control was between 79° C. to 81° C. (FIG.4B). Melting curve analysis also showed that the mismatch primersproduced a product with Tm of 87.6° C. from undiluted cDNA whilst the Tmfor 10⁻¹ until 10⁻⁵ diluted cDNA and negative control was between 76° C.to 78° C. (FIG. 4D). A standard curve derived from 10-fold diluted cDNAof vaccine IBDV strain showed a linear relationship was observed betweenthe amount of input cDNA and the Ct value. The regression equation wasCt=5.1279 (log 10 dilution)+19.433 and R²=0.9809 (FIG. 4E).

FIG. 5 shows agarose gel electrophoresis showing the specificity anddetection limit of the PCR assay for cDNA from very virulent strainUPM94/273. The cDNA was diluted 10-fold from 10⁰ (7,700 ng/μl) to 10⁻⁵(0.077 ng/μl) and used in amplification with match and mismatch primercombinations. Lane 1, 100 bp marker (Promega, USA); Lane 2, undilutedcDNA; Lane 3, 10⁻¹ diluted cDNA; Lane 4, 10⁻² diluted cDNA; Lane 5, 10⁻³diluted cDNA; Lane 6, 10⁻⁴ diluted cDNA; Lane 7, 10⁻⁵ diluted cDNA; Lane8, non-template negative control. Specific amplification of PCR productof the expected size 316 bp was observed from 10⁰ to 10⁻³ diluted cDNAwith match primer combination only (arrow) (FIG. 5A). A faintnonspecific amplification was observed from amplification of undilutedcDNA sample using mismatch primer (FIG. 5B). No specific amplificationwas observed from the serially diluted cDNA and non-template negativecontrol when amplification was performed using mismatch primercombination (FIG. 5B).

FIG. 6 shows agarose gel electrophoresis showing the specificity anddetection limit of the PCR assay for cDNA from vaccine strain D78. ThecDNA was diluted 10-fold from 10⁰ (6,600 ng/μl) to 10⁻⁵ (0.066 ng/μl)and used in amplification with match and mismatch primer combinations.Lane 1, 100 bp marker (Promega, USA); Lane 2, undiluted cDNA; Lane 3,10⁻¹ diluted cDNA; Lane 4, 10⁻² diluted cDNA; Lane 5, 10⁻³ diluted cDNA;Lane 6, 10⁻⁴ diluted cDNA; Lane 7, 10⁻⁵ diluted cDNA; Lane 8,non-template negative control. Specific amplification (arrow) wasobserved from 10⁰ to 10⁻³ diluted cDNA with match primer combinationonly (FIG. 6A). A faint band of the nonspecific amplification was alsoobserved from amplification of undiluted cDNA sample using mismatchprimer (FIG. 6B). No specific amplification was observed from theserially diluted cDNA and non-template negative control whenamplification was performed using mismatch primer combination (FIG. 6B).

FIG. 7 depicts the performance of the real-time in detecting specificamplification of vvIBDV (UPM97/61 and UPM94/273) and attenuated vaccine(D78, LZD, TAD and IBDVAC) strains.

FIG. 8 shows agarose gel electrophoresis showing the specificity of thereal-time PCR assay in detecting vaccine strains; TAD, LZD and IBDVACusing both match and mismatch primer combinations. The real-time PCR wasperformed using 4,000 ng/μl of cDNA. Lane 1, 100 bp marker (Promega,USA); Lane 2, TAD Gumboro with mismatch primer; Lane 3, Delvax LZD withmismatch primer; Lane 4, IBDVAC with mismatch primer; Lane 5, TADGumboro with match primer; Lane 6, Delvax LZD with match primer and Lane7, IBDVAC with match primer. PCR product with the expected size 316 bp(arrow) was observed only with match primer combination.

FIG. 9 depicts the specificity of real-time PCR using IF & IVIR and IF &RCLA primer combinations on control uninfected tissue samples. Nospecific amplifications were detected using IF & IVIR and IF & RCLAprimers combinations on ˜4,000 ng/μl of total RNA extracted from bursa,thymus and cecal tonsil of control uninfected SPF-chickens. As expectedthe positive control sample, UPM94/273 showed specific amplificationusing match primer IF and IVIR. No amplification from the negativecontrol tissue and the Tm values ranged between 72.0° C. to 79.6° C.

FIG. 10 shows nucleotide sequences (316 bp) of UPM94/273, UPM97/61, D78,LZD, TAD and IBDVAC used in this study. The nucleotide sequences ofprimers IF, IVIR and RCLA are also given (SEQ ID NOs:3-5). Thenucleotide sequences encompassed from position 1835 to 2133 of the VP4region.

DETAILED DESCRIPTION OF THE INVENTION

The field of the invention is detection of IBDV in homogenate tissuesamples using different primer combinations. Total RNA from test sampleis reverse transcribed using a pair of conserved primer. The RT productis amplified using different primer combinations where by, differentIBDV strains can be differentiated based the detection of Ct and Tmvalues. Based on these principals, a pair of primer, primers FVVC & RVVCthat hybridized to the conserved region are designed and used in RTreaction.

The RT products are then used as template in cDNA amplification using 2pairs of primers whereby only the sequences of the reverse primers aredifferent depending on the strains of IBDV; primers IF & IVIR andprimers IF & RCLA each are specific to very virulent and vaccine strainsof IBDV, respectively. Since the concentration of cDNA influence thespecificity of the assay, the optimum of cDNA concentration isoptimized. In addition, other parameters including concentration oftotal RNA, primers, MgCl₂, SYBR Green I, PCR profiles andstandardization of the fluorescence threshold level of the real-time PCRmachine were also optimized.

Hence, for differentiation of very virulent from vaccine strains of IBDVprimer IF & IVIR and primers IF & RCLA are used. A PCR amplificationproduct from very virulent strain IBDV has an early amplification (Ctvalue between 19 to 28 and Tm between 86 to 88° C.) and lateamplification (Ct value>29 and Tm<82° C.) or no amplification (Ct value0 and Tm<82° C.), respectively. The invention describes the developmentof a novel SYBR Green I based real-time PCR method for the detection ofvery virulent and vaccine strains of IBDV based on detection ofsignatory Ct and Tm values.

Thus, this invention also includes novel real-time PCR-based assayswhich do not require size determination of the PCR amplification productto confirm the specific amplification of the IBDV target nucleic acidsequence. Therefore, the invention includes simple format assays whichobviate the need for complex molecular biology techniques such asrestriction enzyme digestion and sequencing to confirm that theamplification product is, indeed, of IBDV strains of very virulent orvaccine strains. The methods of the invention are, therefore, less proneto operator error, faster, and may be fully automated. In describing andclaiming the present invention, the following terminology will be usedin accordance with the definitions set out below.

As used herein, “nucleic acid,” “RNA,” “cDNA” and similar terms alsoinclude nucleic acid analogs, i.e. analogs having other than aphosphodiester backbone. For example, the so-called “peptide nucleicacids,” which are known in the art and have peptide bonds instead ofphosphodiester bonds in the backbone, are considered within the scope ofthe present invention.

The term “very virulent” strain of IBDV means samples containing IBDVthat associated with high mortality in chickens and with the followingcharacteristic amino acid residues at the positions 222 (alanine), 242(isoleucine), 256 (isoleucine) and 294 (isoleucine) of the VP2 region,at the positions 680 (tyrosine), 685 (asparagine), 715 (serine) and 751(aspartate) of the VP4 region and at the positions 990 (valine) and 1005(alanine) of the VP3 region. Typically, a very virulent IBDV usuallyassociated with mortality up to 100% in SPF chickens, 25% mortality inbroilers and 60% in layers.

The term “vaccine” strain of IBDV means samples containing IBDV that donot cause mortality in non-vaccinated commercial chickens and with thefollowing characteristic amino acid residues at VP2 region; 253(histidine), 279 (asparagine), and 284 (threonine) and associated withone or more changes at the serine residues of the heptapeptide regionSWSASGS (SEQ ID NO:6) at the position 326 to 332. A vaccine strain alsohas characteristic amino acid residues at the positions 680 (cysteine),685 (lysine), 715 (proline) and 751 (histidine) of the VP4 region and atthe positions 990 (alanine) and 1005 (threonine) of the VP3 region.Typically, a vaccine strain is an attenuated classical IBDV derived fromrepeated passages in embryonated eggs and/or cell cultures.

As used herein, “effective amount” means an amount sufficient to producea selected effect. For example, an effective amount of cDNA is an amountsufficient to amplify a segment of nucleic acid by PCR provided that aDNA polymerase, buffer, template, and other conditions, includingtemperature conditions, known in the art to be necessary for practicingPCR are also provided.

The term “test sample” as used herein, means anything suspected ofcontaining a target sequence. The test sample can be derived from anybiological source and can be used (i) directly as obtained from thesource or (ii) following a pre-treatment to modify the character of thesample. The pre-treatment that can be applied for example, disruptingcells, preparing liquids from solid materials, diluting viscous fluids,filtering liquids, concentrating liquids, inactivating interferingcomponents, adding reagents, purifying nucleic acids, and the like.Typically, the test sample will be derived from bursal tissue samples.

A “target sequence” as used herein means a nucleic acid sequence that isdetected, amplified, or otherwise is complementary to one of the primersherein provided.

A “match primer” as used herein means the nucleotide sequence of primerwith at least the last 3 nucleotide sequences at the 3′end conserved tothe template sequences. Typically, a match primer will has a conservednucleotide sequences with template sequences.

A “mismatch primer” as used herein means the nucleotide sequence ofprimer with at least the last 1 nucleotide sequence at the 3′end differto the template. Typically, a mismatch primer will has 1 to 3 nucleotidevariation at the 3′end with the template sequences.

The term “bp” means base pair.

The term “signatory Ct and Tm values” means consistent readings thatgive selected or desirable effect; to distinguish different strains ofIBDV, i.e., very virulent from vaccine strain. Typically, a match primerand template combination will give an early amplification (Ct valuebetween 19 to 28 and Tm between 86° C. to 88° C.) and late amplification(Ct value>29 and Tm<82° C.) or no amplification (Ct value 0 and Tm<82°C.). A mismatch primer and template combination will give lateamplification (Ct value>29 and Tm<82° C.) or no amplification (Ct value0 and Tm<82° C.).

DETAILED EXAMPLE OF APPLICATION Isolation of Very Virulent and VaccineStrains

A total of 6 IBDV which include 2 field isolates of very virulentstrains (UPM97/61 and UPM94/273) and 4 vaccine strains (Table 1) wereused. The very virulent viruses were in the form of bursal homogenatewhilst the vaccine strains were distilled in 8 to 10 ml of deionisedwater and used for RNA extraction.

TABLE 1 Infectious bursal disease viruses used in the study IsolatesStrains UPM97/61 very virulent UPM94/273 very virulent D78 vaccine TADGumboro vaccine Delvax Gumboro LZD vaccine IBDVAC vaccine

Primer Design

Five different primers were used in this study (Table 2). All theprimers were designed with the aid of Primer Premier 5.0 software. Theprimers were designed based on the following criteria for real-time PCR;primers should be designed to amplify short amplicons. Minimum length ofthe amplicons should be 80 bp and not exceed 400 bp. Ideally, primersshould have about 50% of G/C content. The nucleotide difference shouldbe at the 3′end region of the primers and the region should have no morethan two G and/or C bases.

TABLE 2 Primers used for amplification of different strains of IBDV. Thenucleotide variations between the primers are indicated in bold. PrimerSequence (5′-3′) Positions^(a) SEQ ID NO FVVC^(d) AGA GGG TGC CAC GCTATT 1662-1679 SEQ ID NO: 1 RVVC^(d) GGT ACT GGC GTC CTG CAT T 2255-2237SEQ ID NO: 2 IF^(d) ATG CTC CAG ATG GGG TAC TTC 1835-1855 SEQ ID NO: 3IVIR^(b) TTG GAC CCG GTG TTC ACG 2150-2133 SEQ ID NO: 4 RCLA^(c) TTG GGCCCG GTG TTT ACA 2150-2133 SEQ ID NO: 5 ^(a)The numbering of thenucleotide position based on Bayliss et al. (1990). ^(b)The sequences ofthe primer was conserved when compared to very virulent strains,UPM97/61 (AF247006), UPM94/273 (AF527039), OKYM (D49706), UK661(X92760), IBDKS (L42284), D6948 (AF240686), BD3/99 (AF362776), Tasik94(AF322444), Chinju (AF508176), HK46 (AF092943), SH95 (AF13474), Gx (AY444873), SDH1 (AY323952) and TO9 (AY099456). ^(c)The sequences of theprimer was conserved when compared to vaccine (attenuated) strains, D78(AF499929), Cu-1M (AF362771), P2 (X84034), CT (AJ310185), CEF94(AF194428), PBG-98 (D00868), JD1 (AF321055), HZ-2 (AF321054) and Edgar(AF462026). ^(d)The sequences of the primers were conserved whencompared to both very virulent and attenuated vaccine strains of IBDV asstated above.

The primers FVVC and RVVC were designed from the conserved region of VP4of both very virulent and vaccine strains to generate a 593 bp product.Similar to primers FVVC & RVVC, primer IF was also designed from theconserved region of VP4 of both very virulent and attenuated vaccinestrains whilst primers IVIR and RCLA were designed based on conservedsequences of very virulent and attenuated vaccine strains, respectively,and expected to amplify a 316 bp product.

Primers IVIR and RCLA differed by 3 nucleotide differences at positions2133, 2136 and 2146 (Table 2). The relationship between the primercombinations and IBDV isolates as template is indicated in Table 3. Theprimers IF and IVIR were considered as match primer combination for veryvirulent strains but as mismatch primer combination for vaccine strains.Meanwhile, primers IF and RCLA were considered as match primercombination for vaccine strains but as mismatch primer combination forvery virulent strains.

TABLE 3 Primer combinations and their relationship to template (IBDVisolates) used in real-time PCR. Relationship between primer Primercombinations Isolates Origin Strains combinations^(a) to IBDV UPM97/61UPM very IF & IVIR Match virulent IF & RCLA Mismatch UPM94/273 UPM veryIF & IVIR Match virulent IF & RCLA Mismatch D78 Intervet vaccine IF &IVIR Mismatch IF & RCLA Match TAD Gumboro Lohman vaccine IF & IVIRMismatch IF & RCLA Match Delvax Mycofarm vaccine IF & IVIR MismatchGumboro LZD IF & RCLA Match IBDVAC Veterinary vaccine IF & IVIR MismatchResearch IF & RCLA Match Institute ^(a)The forward primer (IF) isconserved whilst the reverse primers (IVIR and RCLA) varies depending onthe strains of IBDV.

As shown in FIG. 1, the nucleotide difference at position 2146associated with an amino acid substitution at position 715.

RNA Extraction. Total RNA was extracted from the prepared biologicalmaterial using Tri Reagent® (Life Technologies, USA) following themethod described by the manufacturer.

Reverse Transcriptase

The extracted RNA was transcribed into cDNA using primers FVVC and RVVC.In this study, two tubes format of real-time PCR was carried out. Theoptimum conditions of the PCR reaction and programs were optimized usingIBDV strains, UPM94/273 and D78 each represent the very virulent andvaccine strains, respectively. Briefly, a premix reaction containing5,000 ng/μl to 13,000 ng/μl of total RNA, 25 ρmole of primers FVVC &RVVC, 1 μl of DMSO (v/v) in a 10 μl volume was incubated at 99° C. for 5mins. The premix reaction was reverse transcribed at 42° C. for 1 hourin a final volume of 20 μl containing 2× of reaction buffer (Promega,USA), 2 μl of 10 mM dNTP mixture, 5.0 U of AMV reverse transcriptase, 20U of recombinant Rnasin ribonuclease inhibitor. The reaction was thendenatured at 99° C. for 1 min to inactivate the reverse transcriptase.The cDNA was then chilled on ice for 5 mins and was used immediately orstored at −80° C.

Determination of the RNA and cDNA Concentration and Purity

The concentration and purity of the extracted total RNA and cDNA weremeasured at the wavelength of 260 nm and 280 nm using aspectrophotometer.

Optimization of the Real-Time PCR

The condition of the real-time PCR was optimized by using differentconcentration of cDNA and SYBR Green I. Briefly, cDNA (undiluted to1:10⁵ dilution) was used as template was made in 50 μl volume with thefollowing ingredients; 3.0 mM MgCl₂, 2 μl of 10 mM dNTP mixture, 25pmole/μl of each primer (Primer IF & IVIR and Primer IF & RCLA), 2.5 Uof Taq DNA polymerase (Promega, USA), 1 μl of SYBR Green 1 dye diluted1:10³ (Molecular Probe, Eugene, USA) in deionised distilled water(Molecular Probes, USA), 0.8× reaction buffer and cDNA template inlow-profile 0.2 ml tube stripes (MJ Research, USA).

The PCR reactions and conditions that amplified very virulent andvaccine strain, UPM94/273 and D78, respectively, was optimized. It wasfound that the optimum concentration of SYBR Green I as labeling dye was1 μl of 1:10³ diluted stocks. Real-time PCR profiles obtained from 2 μlof 1:10⁴ diluted SYBR Green I stock is not consistent (data not shown).

The amplification was performed in DNA Engine Opticon™ System (MJResearch, USA). No template control and tissue samples (bursa, thymusand ceacal tonsil) of uninfected SPF-chickens were used as negativecontrol. PCR was performed with these conditions; 95° C. for 5 min thenfollowed by 33 cycles of 94° C. for 30 sec, 60° C. for 20 sec and 72° C.for 40 sec then allowed the reaction to be incubated 82° C. to 85° C.before the fluorescence reading was taken. The fluorescence thresholdlimit of the DNA Engine Opticon™ System was set at 0.01.

A real-time PCR assay is also optimized by the number of cycle. It wasfound that amplification of mismatch PCR product occurred after 33cycles. As shown in FIG. 2, the melting temperature (Tm) of the mismatchproduct was 86° C. to 87° C. whilst the Tm for the dimer product was 77°C.

Based on agarose gel electrophoresis (FIG. 2), a faint band of theexpected size, 316 bp was observed with the formation of nonspecificdimer product. Lane M, 100 bp marker (Promega, USA), Lane 2, 3, 4 aretriplicate of D78 with primer IF and IVIR.

Melting Curve Analysis

Upon completion of the amplification, the specificity of the amplifiedproduct was confirmed by melting curve analysis whereby the reaction wasincubated by raising the incubation temperature from 72° C. to 99° C. in0.4° C. increments with a hold of 1 second at each increment. The SYBRGreen I fluorescence (F) was measured continuously during the heatingperiod and the signal was plotted against temperature (T) to produce amelting curve for each sample. The melting peaks were then generated byplotting the negative derivative of the fluorescence over temperatureversus the temperature (−dF/dT versus T). Since SYBR Green I dye bindsto any dsDNA product, specificity and the absence of non-specificamplification were determined by determining the Tm of the non-specificproduct.

Development of the Real-Time PCR to Detect Very Virulent and VaccineStrains of IBDV

After establishing the optimum condition of the real-time PCR, the assaywas performed on very virulent and attenuated vaccine strains, UPM94/273and D78, respectively. The cDNA obtained from both strains were seriallydiluted and used as template in PCR using both match and mismatch primercombinations. FIG. 3 comprising FIGS. 3A, 3B, 3C, 3D and 3E depictingthe amplification from 10-fold dilutions from 10⁰ to 10⁻⁵ of cDNAtemplate of very virulent strain, UPM94/273 using match primer (IF &IVIR) and mismatch (IF & RCLA) primer combinations.

The RT product was generated from 12,000 ng/μl of total RNA.Amplification of specific PCR product using match primer was detectedfrom undiluted (7,700 ng/μl) until 10⁻³ diluted (7.7 ng/μl) cDNAconcentrations (FIG. 3A). However, amplification using mismatch primerwas detected only from undiluted cDNA (FIG. 3C). The specificity of theamplification was also analyzed using melting curve analysis.

The melting temperature (Tm) of the amplicons obtained from undilutedcDNA until 10⁻³ diluted cDNA using match primer combinations was between87.2° C. to 87.6° C. whilst the Tm for 10⁻⁴ to 10⁻⁵ diluted cDNA andnegative control ranged from 78.0° C. to 78.4° C. (FIG. 3B). The meltingcurve analysis also showed that the mismatch primers produced a productwith Tm of 87.6° C. from undiluted cDNA whilst the Tm for 10⁻¹ until10⁻⁵ diluted cDNA and negative control was 81.2° C. to 81.6° C. (FIG.3D) (Table 4). Hence, the detection of specific amplification based ondetection on Ct and Tm analysis was up to 10⁻³ diluted cDNA (7.7 ng/μl).

The correlation between the concentration of the cDNA and Ct values wasanalyzed by plotting a standard curve. As shown in FIG. 3E, Ct valuescan only be detected from amplification of undiluted to 10⁻⁴ dilutedcDNA using match primer combination with a linear relationship betweenthe amount of input cDNA and the Ct value from the amplification. Theregression equation was Ct=3.7765 (log 10 dilution)+16.393 and R²=0.999(FIG. 3E). The Ct and Tm values obtained from amplification of seriallydiluted cDNA of very virulent IBDV strain UPM94/273 are summarized inTable 4 and 7, respectively.

The performance of the real-time PCR in detecting vaccine strain IBDVwas also analyzed and showed very similar as found for very virulentstrain UPM94/273 (FIG. 4A to 4E). The RT product was generated from6,500 to 7,000 ng/μl of total RNA. Amplification of PCR product usingmatch primer was detected from undiluted (6,600 ng/μl) until 10⁻³diluted (6.6 ng/μl) cDNA concentration (FIG. 4A). However, amplificationusing mismatch primer was detected only from undiluted cDNA (FIG. 4C).

However, the Tm of the amplicons obtained from match primer combinationswas between 86.4° C. to 86.8° C. whilst the Tm for 10⁻⁴ and 10⁻⁵ dilutedcDNA as well as negative control ranged from 80.4° C. to 81.6° C. (FIG.4B). The melting curve analysis also showed that the mismatch primersproduced a product with Tm of 87.6° C. from undiluted cDNA whilst the Tmfor 10⁻¹ until 10⁻⁵ diluted cDNA and negative control was 77.2° C. to77.6° C. (FIG. 4D) (Table 5). As shown in Table 5, Ct values can only bedetected from amplification of undiluted to 10⁻⁴ diluted cDNA usingmatch primers. However, the detection of specific amplification based ondetection on Ct and Tm analysis was up to 10⁻³ diluted cDNA (6.6 ng/μl).A standard curve line was generated from amplification of the seriallydiluted vaccine strain, D78 by using match primer combination. As shownin FIG. 4E, a linear standard curve line was also observed between theserially diluted cDNA and the Ct value with the regression equation ofCt=5.1279 (log 10 dilution)+19.433 and R²=0.9809 (FIG. 4E). The Ct andTm values obtained from amplification of serially diluted cDNA ofvaccine IBDV strain D78 are summarized in Table 5 and 8, respectively.

TABLE 4 Threshold cycle (C_(t)) and melting temperature (Tm) values ofamplification of serially diluted cDNA of vvIBDV UPM94/273 using matchand mismatch primer combinations Amplification using different primercombinations^(a) Primer IF & Primer IF & IVIR (Match) RCLA (Mismatch)cDNA dilution/ Tm Tm concentration Ct value value (° C.) Ct value value(° C.) undiluted/7,700 ng/μl 16.40 87.6 19.59 87.6 1:10¹/770 ng/μl 20.4187.6 0 81.6 1:10²/77 ng/μl 24.10 87.6 0 81.6 1:10³/7.7 ng/μl 27.45 87.20 81.6 1:10⁴/0.77 ng/μl 31.70 78.0 0 81.2 1:10⁵/0.077 ng/μl 0 78.4 081.6 ^(a)The real-time PCR was performed using 1 μl of SYBR Green I(diluted 1:10³) as labeling dye and the fluorescence threshold limit setat 0.01.

TABLE 5 Threshold cycle (Ct) and melting temperature (Tm) values ofamplification of serially diluted cDNA of vaccine IBDV D78 using matchand mismatch primer combinations Amplification using different primercombinations^(a) Primer IF & Primer IF & RCLA (Match) IVIR (Mismatch)cDNA dilution/ Tm Tm concentration Ct value value (° C.) Ct value value(° C.) undiluted/6,660 ng/μl 19.43 86.8 19.42 87.2 1:10¹/660 ng/μl 24.2786.8 0 77.2 1:10²/66 ng/μl 27.72 86.8 0 77.2 1:10³/6.6 ng/μl 33.34 86.40 77.6 1:10⁴/0.66 ng/μl 40.53 81.6 0 77.6 1:10⁵/0.066 ng/μl 0 80.4 077.2 ^(a)The real-time PCR was performed using 1 μl of SYBR Green I(diluted 1:10³) as labeling dye and the fluorescence threshold limit wasset at 0.01.

Agarose Gel Electrophoresis Analysis

Amplification of the real-time PCR products was also verified on 1.7%agarose gel in TAE buffer. The size of the product was estimated using100 bp DNA ladder (Promega, USA). The specificity and detection limit ofthe PCR using different primer combinations in detecting very virulentand vaccine strains IBDV were also confirmed by agarose gelelectrophoresis. Specific amplification of PCR product of the expectedsize 316 bp was observed from 10⁰ to 10⁻³ diluted cDNA of UPM94/273 withmatch primer combination only (FIG. 5A). Nonspecific product wasobserved only from amplification of undiluted cDNA sample. In the caseof mismatch primer combination, no amplification was detected except fornonspecific PCR products (˜400 base pair and ˜600 base pair) wereobserved from undiluted cDNA sample (FIG. 5B).

Similar results were obtained when the PCR products from D78 wereanalyzed on 1.7% agarose gel. A PCR product of the expected size (316bp) was observed from 10⁰ to 10⁻³ diluted cDNA using match primercombination only (FIG. 6A). When the serially diluted cDNA were testedusing mismatch primer combinations, nonspecific amplification wasdetected only from the undiluted cDNA whilst, no amplification wasdetected from the diluted cDNA samples (FIG. 6B).

Evaluation of the Real-Time PCR

After establishing the real-time PCR condition and the detection limitsfor the detection of very virulent and vaccine strains, UPM94/273 andD78, respectively, the assay was also performed on other IBDV isolatesas listed in Table 1. The real-time PCR was performed using both matchand mismatch primer combinations and the total RNA concentration rangingfrom 4,500 to 12,800 ng/μl. The real-time PCR was also tested usingtissue samples such as bursa, thymus and ceacal tonsil of controluninfected SPF-chickens. As shown in Table 6, no Ct values weregenerated from amplification of the control negative tissue samples andthe Tm values were in the ranged of 72.0° C. to 79.6° C.

However, as expected the match primer combinations amplified theUPM94/273 sample. No specific amplifications were detected from thosetissue samples using both match and mismatch primers (FIG. 9). However,the cDNA concentration was set at 4000 ng/μl. The amplification profilesof the real-time PCR assay for the amplification of IBDV strainsUPM97/61, UPM94/273, D78, Delvax LZD, TAD Gumboro and IBDVAC were shownin FIGS. 7A, 7B, 7C, 7D, 7E and 7F, respectively. Regardless of the IBDVisolates, the specific amplification was detected only from match primercombination with the Ct value of the amplified products ranged from 19to 28 and the Tm of the amplified products ranged from 86° C. to 88° C.for both cDNA obtained from very virulent and vaccine strains.

The very virulent strains, UPM94/273 and UPM97/61 were amplified onlywith the match primer (primer IF & IVIR) whilst the vaccine strains,D78, Delvax LZD, TAD Gumboro and IBDVAC were amplified only with matchprimer (primer IF & RCLA). No amplification with Ct value 0 was detectedfor amplification of the IBDV using mismatch primer combinations.

The specific amplification of the vaccine strains, Delvax LZD, TADGumboro and IBDVAC using match and mismatch primer combination were alsoverified using 1.7% agarose gel. As shown in FIG. 8, specificamplification of PCR product of the expected size (316 bp) was detectedonly from match primer but not mismatch primer combinations.

TABLE 6 Threshold cycle (Ct) and melting temperature (Tm) values ofamplification of negative control samples. Primer IF & IVIR Primer IF &RCLA Samples^(a) Ct Tm (° C.) Ct Tm (° C.) UPM94/273 (positive control)19.158 88.0 None 72.0 Bursa None 78.8 None 72.0 Thymus None 79.6 None79.6 Cecal tonsil None 79.2 None 79.6 ^(a)The concentration of theundiluted cDNA was 4000 ng/μl.

TABLE 7 Detection of signatory threshold cycle (Ct) values of veryvirulent and vaccine strains IBDV using different primer combinations.Threshold cycle (Ct) value^(a) Isolates Strains Primer IF & IVIR PrimerIF & RCLA UPM97/61 very virulent 19 to 28 >29 or 0 UPM94/273 veryvirulent 19 to 28 >29 or 0 D78 vaccine >29 or 0 19 to 28 TAD Gumborovaccine >29 or 0 19 to 28 Delvax Gumboro vaccine >29 or 0 19 to 28 LZDIBDVAC vaccine >29 or 0 19 to 28 ^(a)The real-time PCR was performedusing 4000 ng/μl of cDNA, 1 μl of SYBR Green I (diluted 1:10³) aslabeling dye and the fluorescence threshold limit was set at 0.01.

The SYBR Green I based real-time PCR detects both very virulent andvaccine strain IBDV as detected based on the Ct and Tm values. However,the detection based on Tm has lower CV value, 0.87 compared to Ct with aCV of 9.58 (Table 9). This suggests that Tm is a consistent parameter indetecting specific amplification. The high CV for Ct values are expectedsince the absolute quantity of the viral RNA in the samples may bedifferent. It has been showed that the initial copy number of targetedsequences in the template significantly influencing the Ct values(Mackay et al., 2002).

TABLE 8 Detection of signatory melting temperature (Tm) values from veryvirulent and vaccine strains IBDV using different primer combinations.Melting temperature (Tm) values (° C.)^(a) Isolates Strains Primer IF &IVIR Primer IF & RCLA UPM97/61 very virulent 86 to 88 <80 UPM94/273 veryvirulent 86 to 88 <80 D78 vaccine <80 86 to 88 TAD Gumboro vaccine <8086 to 88 Delvax Gumboro vaccine <80 86 to 88 LZD IBDVAC vaccine <80 86to 88 ^(a)The real-time PCR was performed using 4000 ng/μl of cDNA, 1 μlof SYBR Green I (diluted 1:10³) as labeling dye and the fluorescencethreshold limit set at 0.01.

TABLE 9 Intra-assay variation of Ct and Tm values of real-time PCR usingmatch primer combination in detecting very virulent and vaccine strainsof IBDV Threshold cycle (Ct) Melting temperature (Tm) Interval RangeMean ± SD CV Interval Range Mean ± SD CV 19.57-25.66 6.09 22.775 ± 2.189.58 85.6-87.6 2.00 86.8 ± 0.759 0.87 CV = coefficient variations SD =standard deviations

Purification of PCR Products

The expected PCR products (˜593 bp) generated from primers, FVVC andRVVC were purified by using GENECLEAN (BIO 101, USA) following themanufacturer's instructions. Briefly, the PCR products were cut from theagarose gel by scalpel blade and the weight of the each excided band wasmeasured. Three volumes (three times the weight of the gel) of SodiumIodine (NaI) was added and incubated at 45° C.-55° C. water bath for 5min until all of the gel dissolved. The glass milk (containing insolublesilica matrix) was vortex vigorously for 1 min. Approximately 5 μl ofglass milk was added for each tube (solution containing 5 μg or lessDNA) and mixed well, incubated for 5 min at room temperature and mixedevery 1-2 min to allow binding of the DNA to the silica matrix.

The silica matrix with the bound DNA was then pelleted in amicrocentifuge for 5 secs at full speed. The pellet was then washedthree times with 10-15 volume (approximately 500 μl) New Wash buffer.The pellet was resuspended in the wash by pipetting back and forth whiledigging into the pellet with the pipet tip and centrifuged at 13000 rpmfor 5 sec at 4° C. After the third wash, the last bit of liquid wasremoved by centrifuged the tube again for a few seconds, the pellet wasallowed to dry at RT (5 to 10 min). The DNA was eluted from the glassmilk by resuspending the white pellet with 10 μl of sterile dH2O,centrifuged at 13000 rpm for 1 min. The yield of the eluted DNA wasestimated by agarose gel electrophoresis.

DNA Sequencing

The yield of the eluted DNA was estimated by agarose gelelectrophoresis. The purified products obtained from PCR amplificationfrom very virulent and vaccine strains were sequenced using primers IF &IVIR and primers IF & RCLA, respectively (Table 2). Each PCR product wassequenced twice from both directions. The sequencing was carried outusing ABI PRISM® BigDye Terminator Cycle Sequencing Ready Reaction Kitv2.0 (Perkin Elmer, USA) in an automated DNA sequencer (ABI PRISM® 377DNA Sequencer) following the instructions supplied by the manufacturer.The cycle sequencing was conducted with the following thermal cycleprofiles; 30 cycles, each with 96° C. for 10 seconds, 50° C. for 5seconds, and 60° C. for 4 minutes.

Sequence Analysis of the PCR Amplified Product

As shown in FIG. 10, a total of 316 bp sequences encompassing the VP4gene from position 1835 to 2133 of the PCR products of UPM94/273,UPM97/61, D78, Delvax LZD, TAD Gumboro and IBDVAC were characterized.The nucleotide sequences of primer FVVC, VVC, IF, IVIR and RCLA werealso given. The sequences of the match primers were conserved whencompared to the respective isolates except for the vaccine strain,IBDVAC. IBDVAC has two nucleotide variations each on primer IF and RCLA.At primer IF, the nucleotide variation is at position 1851 from A to Twhilst at primer RCLA, the variation is at position 2141 from C to T(FIG. 10).

1. A diagnostic assay kit adapted or assembled to perform a method ofdetecting Infectious Bursal Disease Virus (IBDV) strains in a chicken orother bird sample, comprising: at least one oligonucleotide primer pair,wherein said oligonucleotide primer pair consists of SEQ ID NO:1 and SEQID NO:2.
 2. The diagnostic assay kit of claim 1, further comprisingreagents for reverse transcribing into cDNA an RNA sample from a chickenor other bird that is infected with IBDV strains to be distinguished. 3.The diagnostic assay kit of claim 1, further comprising reagents foramplifying said IBDV strains by means of a polymerase chain reaction(PCR) using the oligonucleotide primer pair.
 4. The diagnostic assay kitof claim 3, wherein said PCR is real-time (RT)-PCR.
 5. The diagnosticassay kit of claim 3, wherein the PCR produces an amplified product inthe size of 593 base pair (bp).
 6. The diagnostic assay kit of claim 1,further comprising SYBR Green I.
 7. The diagnostic assay kit of claim 1,further comprising reagents for isolating said IBDV strains in thesample.
 8. The diagnostic assay kit of claim 1, wherein one of said IBDVstrains is selected from the group consisting of very virulent strainsUPM97/61 and UPM 94/273.
 9. The diagnostic assay kit of claim 1, whereinone of said IBDV strains is selected from the group consisting ofvaccine strains D78, TAD Gumboro, Delvax Gumboro LZD, and IBDVAC. 10.The diagnostic assay kit of claim 1, further comprising reagents fordiluting cDNA from said IBDV strain(s).
 11. The diagnostic assay kit ofclaim 1, wherein the diagnostic assay kit differentiates between veryvirulent IBDV strains and vaccine IBDV strains.
 12. A diagnostic assaykit adapted or assembled to perform a method of distinguishing betweenInfectious Bursal Disease Virus (IBDV) strains in a chicken or otherbird sample, comprising: at least one oligonucleotide primer pair,wherein said oligonucleotide primer pair consists of SEQ ID NO:3 and SEQID NO:4 or SEQ ID NO:3 and SEQ ID NO:5.
 13. The diagnostic assay kit ofclaim 12, further comprising reagents for reverse transcribing into cDNAan RNA sample from a chicken or other bird that is infected with IBDVstrains to be distinguished.
 14. The diagnostic assay kit of claim 12,further comprising reagents for amplifying said IBDV strains by means ofa polymerase chain reaction (PCR) using the at least one oligonucleotideprimer pair.
 15. The diagnostic assay kit of claim 14, wherein said PCRis real-time (RT)-PCR.
 16. The diagnostic assay kit of claim 14, whereinthe PCR produces an amplified product in the size of 361 base pair (bp).17. The diagnostic assay kit of claim 14, further comprising reagentsfor distinguishing said IBDV strains based on melting temperature (Tm)values of a nucleic acid product of the PCR amplification.
 18. Thediagnostic assay kit of claim 12, further comprising SYBR Green I. 19.The diagnostic assay kit of claim 12, further comprising reagents forisolating said IBDV strains in the sample.
 20. The diagnostic assay kitof claim 12, wherein the diagnostic assay kit distinguishes veryvirulent strain UPM 94/273 and other IBDV strains that have nucleotidesequence complementary to SEQ ID NO:3 and SEQ ID NO:4.
 21. Thediagnostic assay kit of claim 12, wherein the diagnostic assay kitdistinguishes vaccine strain D78 and other IBDV strains that havenucleotide sequence complementary to SEQ ID NO:3 and SEQ ID NO:5. 22.The diagnostic assay kit of claim 12, further comprising reagents fordiluting cDNA from said IBDV strain(s).
 23. The diagnostic assay kit ofclaim 22, wherein said diluted cDNA from very virulent strains isdiluted from at least 100 to 10⁻⁵ fold.
 24. The diagnostic assay kit ofclaim 22, wherein said diluted cDNA from vaccine strains is diluted fromat least 100 to 10⁻⁵ fold.
 25. A diagnostic assay kit adapted orassembled to perform a method for detecting and/or differentiating IBDVin chicken or bird samples, comprising: oligonucleotide primer pairsconsisting of SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4or SEQ ID NO:3 and SEQ ID NO:5.